1. Biofuels
Biofuels represent renewable energy sources from living organisms, such as higher plants, fungi, or bacteria. Photosynthetic life forms capture light energy and subsequently convert it into the free energy of organic compounds based on fixed CO2, using water as the ultimate electron donor. Currently, two major technologies are employed for generating biofuels using phototrophic organisms: first, plant-based biofuel production via fermentation of the plant's sugar content to ethanol and, second, to a much lesser extent, algae-derived biodiesel production through lipid extraction of biomass from large-scale cultures (Angermayr et al., 2009, Curr Opin Biotechnol, 20(3): 257-263).
2. Fatty Acids
Fatty acids are carboxylic acids with hydrocarbon chains of 4 to 36 carbons. In some fatty acids, this chain is fully saturated (meaning contains no double bonds) and unbranched; others contain one (monounsaturated) or more double bonds (polyunsaturated). A few contain three-carbon rings or hydroxyl groups. A simplified nomenclature for these compounds specifies the chain length and number of double bonds, separated by a colon; the 16-carbon saturated palmitic acid is abbreviated 16:0, and the 18-carbon oleic acid, with one double bond, is 18:1. The positions of any double bonds are specified by superscript numbers following Δ (delta); a 20-carbon fatty acid with one double bond between C-9 and C-10 (C-1 being the carboxyl carbon), and another between C-12 and C-13, is designated 20:2 (Δ9,12), for example. The most commonly occurring fatty acids have even numbers of carbon atoms in an unbranched chain of 12 to 24 carbons. The even number of carbons results from the mode of synthesis of these compounds, which involves condensation of acetate (two-carbon) units. (Lehninger et al., Principles of Biochemistry, Vol. 1, Macmillan, 2005).
The position of double bonds in unsaturated fatty acids also is irregular; in most monounsaturated fatty acids, the double bond is between C-9 and C-10 (Δ9), and the other double bonds of polyunsaturated fatty acids are generally Δ12 and Δ15. The double bonds of polyunsaturated fatty acids are almost never conjugated (alternating single and double bonds), but commonly are separated by a methylene group (—CH═CH—CH2—CH═CH—). The physical properties of the fatty acids, and of compounds that contain them, are largely determined by the length and degree of unsaturation of the hydrocarbon chain, i.e., the longer the fatty acyl chain and the fewer the double bonds, the lower the solubility in water. (Lehninger et al., Principles of Biochemistry, Volume 1, Macmillan, 2005).
2.1 Fatty Acid Composition of Algae and Cyanobacteria
Algae are at the base of the trophic ladder of aquatic ecosystems, providing energy and essential nutrients for primary consumers. The major acyl lipid classes in algae are typically phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol), glycolipids (e.g., monogalactosyldiglycerol, digalactosyl glycerol sulfolipids), triacylglycerols, sterol esters, and free fatty acids. Phospholipids are structural constituents of cellular membranes, whereas glycolipids are major components of the thylakoid membrane in chloroplasts. Triacylglycerols produced by some algal species may be present as intracellular storage material and can occur as clearly visible oil droplets. Sterol esters are normally minor lipid constituents in plants (Arts and Wainman, Lipids in Freshwater Ecosystems, Springer, 1998).
Cyanobacterial cells are observed to resemble chloroplasts of eukaryotic plants in terms of membrane structure and glycerolipid composition. There are three types of membrane in the cyanobacterial cells, namely, the plasma membrane, the outer membrane, and the thylakoid membranes. The thylakoid membranes are closed systems and are separated from the plasma membrane. This architecture corresponds to that of the eukaryotic chloroplasts, which has inner and outer envelope membranes and thylakoid membranes. (Murata and Wada, Biochem. J., 1995, 308: 1-8).
The major nonpigment lipids of cyanobacteria have been identified as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglyerol (DGDG), phosphatidylglycerol, and sulfoquinovosyldiacylglycerol. Other lipids observed to occur in lesser amounts include fatty acids, sterols, hydrocarbons, and heterocyst glycolipids (glycosides of long chain diols, triols, and hydroxy acids). Cyanobacteria are not known to contain phosphatidylcholine, -ethanolamine, -serine, -inositol, and diphosphatidylglycerol (cardiolipin) (Ikawa et al., J. Chem. Ecol., 1994, 20: 2429-2436).
2.2 Fatty Acid Biosynthesis
The irreversible formation of malonyl-CoA from acetyl-CoA is catalyzed by acetyl-CoA carboxylase in what is considered to be the first committed step in fatty acid biosynthesis (FIG. 1). Acetyl-CoA carboxylase contains biotin as its prosthetic group, covalently bound in amide linkage to the s-amino group of a Lys residue on one of the three subunits of the enzyme molecule. The carboxyl group, derived from bicarbonate (HCO3-), is first transferred to biotin in an ATP-dependent reaction. The biotinyl group serves as a temporary carrier of CO2, transferring it to acetyl-CoA in the second step to yield malonyl-CoA. (Lehninger et al., Principles of Biochemistry, Volume 1, Macmillan, 2005).
In contrast to other heterotrophic bacteria, such as E. coli, which have to metabolize glucose from media into acetyl-CoA in order to initiate the fatty acid synthesis, in cyanobacteria, the precursor for fatty acid synthesis, i.e., acetyl-CoA, directly comes from the Calvin-Benson cycle which fixes carbon dioxide using energy and reducing power provided by the light reactions of photosynthesis.
The reaction sequence by which the long chains of carbon atoms in fatty acids are assembled consists of four steps: (1) condensation; (2) reduction; (3) dehydration; and (4) reduction. The saturated acyl group produced during this set of reactions is recycled to become the substrate in another condensation with an activated malonyl group. With each passage through the cycle, the fatty acyl chain is extended by two carbons. In many cells, chain elongation terminates when the chain reaches 16 carbons, and the product (palmitate, 16:0) leaves the cycle. The methyl and carboxyl carbon atoms of the acetyl group become C-16 and C-15, respectively, of the palmitate; the rest of the carbon atoms are derived from malonyl-CoA. All of the reactions in the synthetic process are catalyzed by a multienzymatic complex, the fatty acid synthase (Lehninger et al., Principles of Biochemistry, Volume 1, Macmillan, 2005).
2.3. The Elongation Cycle in Fatty Acid Synthesis
Fatty acid synthesis represents a central, conserved process by which acyl chains are produced for utilization in a number of end-products such as biological membranes. The enzyme system, which catalyzes the synthesis of saturated long-chain fatty acids from acetyl CoA, malonyl-CoA, and NADPH, is called the fatty acid synthase (FAS) (FIG. 1). Fatty acid synthases (FASs) can be divided into two classes, type I and II, which are primarily present in eukaryotes and in bacteria and plants respectively. They are characterized by being composed of either large multifunctional polypeptides in the case of type I or consisting of discretely expressed mono-functional proteins in the type II system. (Chan D. and Vogel H, Biochem J., 2010, 430(1):1-19). The fatty acid synthase contains six catalytic activities and contains beta-ketoacyl synthase (KS), acetyl/malonyl transacylase (AT/MT), beta-hydroxyacyl dehydratase (DH), enoyl reductase (ER), beta-ketoacyl reductase (KR), acyl carrier protein (ACP), and thioesterase (TE) (Chirala and Wakil, Lipids, 2004, 39(11):1045-53). It has been shown that the reactions leading to fatty acid synthesis in higher organisms are very much like those of bacteria (Berg et al, Biochemistry, 6th ed., Macillan, 2008).
Fatty acid biosynthesis is initiated by the fatty acid synthase component enzyme acetyltransferase loading the acyl primer, usually acetate, from coenzyme A (CoA) to a specific binding site on fatty acid synthase (FAS). At the end of the process, termination of chain elongation occurs by removing the product from the fatty acid synthase (FAS) either by transesterification to an appropriate acceptor or by hydrolysis. The respective enzymes are usually palmitoyl transferase and thioesterase. The reaction sequence between initiation and termination involves the elongation of enzyme-bound intermediates by several iterative cycles of a distinct set of reaction steps. Each cycle includes (i) malonyl-transacylation from CoA to the enzyme by malonyl transferase; (ii) condensation of acyl-enzyme with enzyme-bound malonate to 3-ketoacyl-enzyme by 3-ketoacyl synthase, (iii) reduction of the 3-keto- to the 3-hydroxyacyl intermediate by ketoacyl reductase, (iv) dehydration of 3-hydroxyacyl enzyme to 2,3-trans-enoate by dehydratase, and, (v) finally, reduction of the enoate to the saturated acyl-enzyme by enoyl reductase. The prosthetic group, 4′-phosphopantetheine, plays a central role in substrate binding, processing of intermediates, and communicating of intermediates between the various catalytic centers of fatty acid synthase (FAS). This cofactor is bound covalently to a specific serine hydroxyl group of the ACP domain or, depending on the FAS system, to the ACP component of FAS. In some bacteria, the iterative sequence of elongation cycles may be interrupted at a chain length of 10 carbons by one cycle involving an intrinsic isomerase converting the 2-trans- into the 3-cis-decenoyl intermediate, which is subsequently not reduced but further elongated to long-chain monounsaturated fatty acids (Schweizer and Hofmann, Microbiol Mol Biol Rev., 2004, 68(3): 501-17).
3. Acyl Carrier Protein (ACP)
The acyl carrier protein (ACP), the cofactor protein that covalently binds fatty acyl intermediates via a phosphopantetheine linker during the synthesis process, is central to fatty acid synthesis. It is a highly conserved protein that carries acyl intermediates during fatty acid synthesis. ACP supplies acyl chains for lipid and lipoic acid synthesis, as well as for quorum sensing, bioluminescence and toxin activation. Furthermore, ACPs or PCPs (peptidyl carrier proteins) also are utilized in polypeptide and non-ribosomal peptide synthesis, which produce important secondary metabolites, such as, the lipopeptide antibiotic daptomycin and the iron-carrying siderophore enterobactin (Chan and Vogel, Biochem. J., 2010, 430:1-19).
In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plants (type II FAS) (Byers and Gong, Biochem Cell Biol., 2007, 85(6): 649-62).
In E. coli, ACP is highly abundant, comprising approximately 0.25% of all soluble proteins and it represents one of four major protein-protein interaction hubs, the others being DNA and RNA polymerases as well as ribosome-associated proteins. In type I FAS systems, ACP is part of large, multi-domain polypeptides that also carry the other protein domains for FA synthesis in a linear fashion. Although the architecture and sequence identity of the type I FAS systems are different from the type II dissociated enzymes, many of the functional units in these complexes are similar. On the other hand, other domains, such as the enoyl reductase and dehydratase enzymes, vary significantly between the type Ia, Ib and II systems (Chan and Vogel, Biochem. J., 2010, 430:1-19).
4. Acyl-ACP Thioesterases
The major termination reaction of fatty acid biosynthesis is catalyzed by acyl-acyl carrier protein (acyl-ACP) thioesterases in eukaryotes. Previous studies have shown that the acyl-ACP thioesterase enzyme terminates acyl elongation of a fatty acyl group by hydrolyzing an acyl group on a fatty acid. In plants, an acyl-ACP thioesterase terminates the acyl elongation process by hydrolysis of the acyl-ACP thioester; free fatty acid then is released from the fatty acid synthase. In E. coli, the long-chain acyl group is transferred directly from ACP to glycerol-3-phosphate by a glycerol-3-phosphate acyltransferase, and free fatty acids normally are not found as intermediates in lipid biosynthesis. As in most other organisms, the major end products of the plant and E. coli fatty acid synthase are usually 16- or 18-carbon fatty acids. Chain length is determined by the 3-ketoacyl-ACP synthases I and II and the glycerol-3-phosphate acyltransferase in E. coli. (Voelker and Davies, J. Bacteriol, 1994, 17: 7320-7327)
4.1. Plant Acyl-ACP Thioesterases
Acyl-ACP thioesterases have been studied extensively in plants. In plants, de novo fatty acid synthesis occurs in the stroma of plastids, where the acyl chains are covalently bound to a soluble acyl carrier protein (ACP) during the extension cycles. Carbon chain elongation can be terminated by transferring the acyl group to glycerol 3-phosphate, thereby retaining it in the plastidial “prokaryotic” lipid biosynthesis pathway. Alternately, specific thioesterases can intercept the prokaryotic pathway by hydrolyzing the newly formed acyl-ACP into free fatty acids and ACP. Subsequently, the free fatty acids exit the plastids by an undetermined mechanism and supply the “eukaryotic” lipid biosynthesis pathway. The latter is located in the endoplasmic reticulum and is responsible for the formation of phospholipids, triglycerides, and other neutral lipids. By catalyzing the first committed step in the eukaryotic lipid biosynthesis pathway in plant cells, acyl-ACP thioesterases play a crucial role in the distribution of de novo synthesized acyl groups between the two pathways (Lohden and Frentzen, Planta, 1988, 176: 506-512; Browse and Somerville, Plant Mol. Biol, 1991, 42: 467-506; and Gibson et al., Plant Cell Environ, 1994, 17: 627-637).
Acyl-ACP thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. There are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB. FatA encodes a C18:1-ACP thioesterase. In contrast, FatB encodes thioesterases preferring acyl-ACPs having saturated acyl groups (Jones et al., Plant Cell, 1995, 7(3):359-71).
Among prokaryotes, acyl groups exiting the dissociable fatty acid synthase are transferred directly from ACP to polar lipids. In contrast, plants must also release sufficient fatty acid from ACP to supply the extraplastidial compartments. Analysis of cloned plant thioesterases suggested that plants possess individual thioesterases with specificity either for C18:1 or for one or more saturated fatty acids (Vance et al., Biochemistry of Lipids, Lipoproteins, and Membranes, Elsevier, 1996). The most prominent thioesterase in most plants has a strong preference for C18:1-ACP, making C18:1 the fatty acid most available for extraplastidial glycerolipid synthesis. Several plant species that produce storage oils containing large amounts of fatty acids having an acyl chain length from 8 to 14 carbons contain thioesterases specific for those acyl chain lengths. By removing acyl groups from ACP prematurely, the medium-chain thioesterase simultaneously prevents further chain elongation and releases fatty acids for triacylglycerol synthesis outside the plastids. Thus, by regulating expression of different thioesterases, plants can both fine tune and radically modify the exported fatty acid pool (Vance et al., Biochemistry of Lipids, Lipoproteins, and Membranes, Elsevier, 1996).
The acyl-ACP hydrolytic specificities of five FatA representatives from three families have been measured in vitro after heterologous expression in E. coli. All FatA thioesterases appeared to be C18:1 specific, with minor activities on C18:0 and C16:0 substrates (Knutzon et al., Plant Physiol, 1992, 100(4):1751-1758; Dormann et al., Plant Physiol., 1994, 104(3): 839-844). In contrast to the conserved nature of FatA, the specificities of FatB enzymes show high variability. The California bay Umbellularia californica (Uc) FatB1 has a strong preference for C12:0 ACP (and a modest preference for C14:0-ACP; Voelker and Davies, J. Bacteriol., 1994, 176(23): 7320-7327). A C. hookeriana thioesterase, encoded by ChFatB2, has been characterized and found to hydrolyze C8:0 and C10:0 ACP; it is the enzyme involved in C8:0 and C10:0 fatty acid production for the storage lipids in seed. In addition, the specificity of a FatB representative from elm (U. americana) seed, Ua FatB1, shows that this enzyme is involved in C10:0 production for the elm storage lipids (80 mol % 10:0; Davies, H., Phytochemistry, 1993, 33:1353-1356). Medium chain-preferring FatB representatives may be common, if not universal, components of the fatty acid synthases specialized for medium-chain production in oilseeds (Jones and Davies, Plant Cell, 1995, 7(3): 359-371). Fat A and Fat B plant acyl-ACP thioesterase enzymes contain a targeting peptide at the N-terminal, which transports the expressed enzymes to the chloroplast where the biosynthesis of fatty acid occurs in plants. When expressed in bacteria, these higher plant thioesterase genes generally are N-terminally truncated in order to remove the chloroplast targeting peptide (Jones and Davies, Plant Cell, 1995, 7(3): 359-371). E. coli and many other prokaryotes do not have acyl-ACP thioesterases (Jones and Davies, Plant Cell, 1995, 7(3): 359-371), but some prokaryotes do have acyl-CoA thioesterases that cleave acyl chains from coenzyme A, which serves as a cofactor in fatty acid degradation.
Current understanding of the role of the acyl-CoA thioesterases in fatty acid metabolism is incomplete. The E. coli acyl-CoA thioesterase TesA (Thioesterase I), for example, is a periplasmic enzyme, but whether it functions in lipid synthesis, recycling, or degradation is unclear. Genes encoding N-terminally truncated TesA or N-terminally truncated TesA variants have been expressed in bacteria. These proteins lack a secretion sequence, and remain in the cytoplasm, where they are able to cleave acyl-ACPs.
For example, U.S. Pat. No. 5,455,167 discloses genes and constructs for expressing genes encoding higher plant acyl-ACP thioesterases, as well as a construct for expressing a gene encoding the Vibrio harveyi LuxD acyl transferase (YP—001448362.1 GI:156977456), belonging to pfam PF02273, in higher plants. PCT Publication No. WO2007/136762 discloses recombinant microorganisms engineered for the fermentative production of fatty acid derivatives, such as, inter alia, fatty alcohols and wax esters, in which the host strain can express a higher plant thioesterase or the E. coli TesA acyl-CoA thioesterase. PCT Publication No. WO2008/100251 describes methods for engineering microorganisms that include genes encoding synthetic cellulosomes to produce hydrocarbon products (which may be, inter alia, alkanes, alkenes, alkynes, dienes, fatty acids, isoprenoids, fatty alcohols, fatty acid esters, polyhydroxyalkanoates, organic acids, and the like). The microorganism that contains one or more exogenous nucleic acid sequence encoding a synthetic cellulosome can also include an exogenous thioesterase gene, such as the E. coli TesA acyl-CoA thioesterase or a plant thioesterase gene, which can be expressed in the host cells.
Other applications disclosing microorganisms, including algae, engineered to express heterologous acyl-ACP thioesterases from higher plants or acyl-CoA thioesterases from E. coli for the production of various compounds, including, inter alia, fatty acids or fatty acid derivatives, include PCT Publication Nos. WO2008/151149, WO2009/076559, and WO2009036385, as well as PCT Publication Nos. WO2009/111513, WO2010/022090, and WO2010/118410.